This research analyzed metabolomic and proteomic differences between participants with gingivitis (>20 bleeding sites) and generally healthy participants (≤3 bleeding sites) at baseline and 4 weeks post stannous fluoride (SnF₂) dentifrice treatment. Sixty-two metabolites were different (p < 0.05) between groups at baseline. Forty cytokines were analyzed using immunoassays and a group of proinflammatory cytokines (IL-1α, IL-1β, TNF-α, SAA, ICAM-1, VCAM-1) was elevated in participants with gingivitis (p < 0.1) versus healthy gingiva at baseline, with C-reactive protein (p < 0.05) being significantly elevated. Proteomic analysis carried out in baseline oral lavage revealed four of the top hits (p < 0.0004) were central-metabolism-related: aldolase A, triosephosphate isomerase, lactate dehydrogenase, and malate dehydrogenase. Enzymatic assays confirmed the proteomic finding that malate dehydrogenase and triosephosphate isomerase activities were elevated in gingivitis samples; SnF₂ dentifrice treatment reduced their activity. Collectively, 20 proteins with the lowest p-values in oral lavage appeared to be indicative of periodontal health, potentially forming the basis to cluster samples into healthy and unhealthy groups. A TLR-ATP biosensor model was established and demonstrated that microbial virulence factors induced the observed changes in oral lavage. Combined findings suggest gingivitis involves upregulation of host cell bioenergetic processes involving enzymatic activity in the glycolysis and citric acid cycle pathways.

This research examined host metabolic processes associated with gingivitis and potential impacts of interventions. First, gingival brush samples from a previously reported clinical study in which participants used an intervention (stannous fluoride (SnF₂) toothpaste/cetylpyridinium chloride rinse/oscillating-rotating electric toothbrush) or control (standard fluoride toothpaste/manual toothbrush) were analyzed with Metabolon metabolomic assessment. Next, hypotheses were generated about mechanisms of metabolic observations and tested in two in vitro models. Human primary blood cells were treated with endotoxins and outer membrane vesicles (OMVs) in the presence of SnF₂. In clinical brush samples, 46 metabolites decreased with intervention at Week 3, including 9- and 13-hydroxyoctadecadienoic acid, citrulline, adenine, and hypoxanthine. Seven metabolites related to nitrogen metabolism and citrulline production were selected for targeted quantification. They were low in health and increased in gingivitis. Targeted quantification demonstrated reduced citrulline, succinate, ornithine and deoxycarnitine (p ≤ 0.001 for all) between baseline and Week 6 with intervention; reductions were greater for intervention vs. control for citrulline, succinate and deoxycarnitine at Week 6 (p ≤ 0.019). In vitro, endotoxin and OMVs increased citrulline and nitric oxide (NO). SnF₂ inhibited elevation of both. Citrulline, deoxycarnitine, succinate, and ornithine were identified as potential novel markers of gingivitis. Elevated NO production is a potential driver of oxidative stress, reduced with intervention.